In an important type of in situ assay, a cell is incubated with a target-specific probe that is attached to a fluorescent dye and subsequently the probe molecules that have bound to the target are detected fluorimetrically. Detection is normally accomplished with a flow cytometer or microscope. Such assays are useful to detect viral nucleic acids, human genes of interest, and specific cellular or viral antigens.
In the first steps of a typical fluorimetric measurement, a cell is exposed to light with a wavelength that the probe dye can absorb. The dye will then emit the light at a longer wavelength. A significant problem, however, is that the light emitted by a probe-treated cell will not exclusively originate from fluorescent probe molecules bound to specific target molecules. Rather some of the light emitted will be from probe molecules that have bound non-specifically; additionally, light will be emitted by cellular molecules.
If the probe molecule has a nucleic acid moiety, probe specificity derives from the fact that a target nucleic acid in the cell has a base sequence that is complementary to that of the probe. Where such complementarity exists, and under conditions where the probe can diffuse into the proximity of the target nucleic acid, the probe and target nucleic acids enter into a double-stranded hybrid molecule relatively efficiently. Independent of such specific reactions, however, the probe molecule will, to some extent, bind to nucleic acid molecules that are not perfectly complementary to the probe, to protein molecules, and to other non-nucleic acid molecules in the cell. Such binding is referred to collectively as non-specific background binding and the light emitted by probes involved in such binding is non-specific background emission.
If the probe has an antibody moiety, specificity derives from the fact that the antibody will react with a specific antigen. Non-specific binding can also occur, however.
In addition to the fluorescent probes in the cell, however, there are cellular molecules which have the capacity to absorb and emit light. Such emitted light is referred to as autofluorescence, and the molecules which produce it are autofluorescing molecules.
The present invention involves the discovery of compounds useful as background reducers in fluorimetric in situ assays.
In addition to the general benefit of increased assay sensitivity that the background reducers provide, there is the additional advantage that, for a given level of sensitivity to be reached, cell treatment procedures such as cell fixation need not be as detrimental to cell integrity as would be the case without the use of the background reducers. As a result, the cells retain their structural integrity for a longer time, as consideration particularly important for flow cytometry.